Dual-wavelength imaging of tumor progression by activatable and targeting near-infrared fluorescent probes in a bioluminescent breast cancer model

Abstract

Bioluminescence imaging (BLI) has shown its appeal as a sensitive technique for in vivo whole body optical imaging. However, the development of injectable tumor-specific near-infrared fluorescent (NIRF) probes makes fluorescence imaging (FLI) a promising alternative to BLI in situations where BLI cannot be used or is unwanted (e.g., spontaneous transgenic tumor models, or syngeneic mice to study immune effects).In this study, we addressed the questions whether it is possible to detect tumor progression using FLI with appropriate sensitivity and how FLI correlates with BLI measurements. In addition, we explored the possibility to simultaneously detect multiple tumor characteristics by dual-wavelength FLI (~700 and ~800 nm) in combination with spectral unmixing. Using a luciferase-expressing 4T1-luc2 mouse breast cancer model and combinations of activatable and targeting NIRF probes, we showed that the activatable NIRF probes (ProSense680 and MMPSense680) and the targeting NIRF probes (IRDye 800CW 2-DG and IRDye 800CW EGF) were either activated by or bound to 4T1-luc2 cells. In vivo, we implanted 4T1-luc2 cells orthotopically in nude mice and were able to follow tumor progression longitudinally both by BLI and dual-wavelength FLI. We were able to reveal different probe signals within the tumor, which co-localized with immuno-staining. Moreover, we observed a linear correlation between the internal BLI signals and the FLI signals obtained from the NIRF probes. Finally, we could detect pulmonary metastases both by BLI and FLI and confirmed their presence histologically.Taken together, these data suggest that dual-wavelength FLI is a feasible approach to simultaneously detect different features of one tumor and to follow tumor progression with appropriate specificity and sensitivity. This study may open up new perspectives for the detection of tumors and metastases in various experimental models and could also have clinical applications, such as image-guided surgery.

Figure 1. Schematic representation of the localization of the signal intensity areas used to calculate TBRs.

TBRs were calculated by dividing the mean fluorescence intensity of four areas, of the same size, located inside the tumor region (1–4) by the mean fluorescence intensity of four areas located approximately 5 mm outside the tumor region (background fluorescence) (5–8).

Figure 2. In vitro NIRF probe activation/binding assays.

4T1-luc2 breast cancer cells were seeded in 96-well plates and incubated with increasing concentrations of Prosense680 or MMPSense680 (A) and IRDye 800CW EGF or IRDye 800CW 2-DG (B) and were subsequently imaged. Intracellular accumulation of Prosense680 (45nM; 24 h incubation) was visualized by confocal microscopy (C). Intracellular accumulation of IRDye 800CW 2-DG (10 µM; 2 h incubation) was visualized by the Nuance multispectral imaging system (D). NIRF probes, cell nuclei and membranes are indicated in red, blue and green, respectively.

Figure 3. In vitro tumor cell detection limit studies.

4T1-luc2 cells were seeded in 96-well plates with various initial densities (39 to 2×10⁴ cells per well). The next day, cells were used for BLI measurement or incubated with either Prosense680 or with IRDye 800CW EGF and then used for FLI measurements. Statistic significance was calculated by determining the difference between optical signals from wells with and without cells. The star indicates the first significant difference. * = P<0.05; *** = P<0.0001.

Figure 4. In vivo tumor cell detection limit studies.

Different numbers of 4T1-luc2 cells were implanted orthotopically into nude mice. 1×10⁵, 5×10⁴, 2.5×10⁴ and 1.25×10⁴ cells were implanted at the upper left, upper right, lower left and lower right MFPs, respectively. BLI measurements at day 1 and 4 are shown in panels A and D. The corresponding IRDye CW800 EGF FLI measurements are shown in panels B and E, respectively. Quantitative analysis of the BLI and FLI measurements using both IRDye CW800 EGF and Prosense680 are shown in panels C and F.

Figure 5. Longitudinal imaging of tumor growth by BLI and FLI.

Representative images of tumor progression by BLI (A–D) and FLI utilizing MMPSense680 (E–H) in combination with IRDye 800CW EGF (I–L) are shown. At the end of the experiment, the lungs of the tumor-bearing mice were re-imaged and co-localizing BLI and FLI signals were observed, indicating the presence of pulmonary metastases (D, H and L).

Figure 6. H&E stained paraffin sections of a primary tumor and of its pulmonary metastases.

Panel A shows a section of a primary 4T1-luc2 breast tumor indicating neoplastic, actively proliferating cells invading the healthy surrounding tissues. Panel B shows subpleural pulmonary metastases of the tumor.

Figure 7. Immunohistological and fluorescent analysis of tumor sections.

The upper panels display sections stained with antibodies against EGFR (A), GLUT-1 (B), MMP-9 (C) and CathepsinB (D). The lower panels show the corresponding sections with FLI signals from animals injected with IRDye 800CW EGF (E), IRDye 800CW 2-DG (F), MMPSense680 (G) or ProSense680 (H). Scale bar = 2 mm.

Figure 8. 4T1-luc2 tumor growth curve indicated by BLI and FLI.

During tumor progression, from day 4 to day 18, there was a steady increase in both BLI and FLI signal intensities.

Figure 9. Correlation plots of BLI against FLI signals obtained longitudinally, utilizing four different NIRF probes.

The Pearson's r values of the NIRF probes were: Prosense680 (r = 0.72, p<0.01), IRDye 800CW 2-DG (r = 0.70, p<0.01), MMPSense680 (r = 0.92, p<0.0001) and IRDye 800CW EGF (r = 0.88, p<0.0001).

Figure 10. TBRs of 4T1-luc2 tumors exploiting four different NIRF probes.

The TBRs, measured on the final day of the experiment, for ProSense680, IRDye 800CW 2-DG, MMPSense680 and IRDye 800CW EGF ranged from 2.29±0.38 to 4.02±0.24.